Conventional therapies have proven to be inadequate in the treatment of many if not most chronic biofilm infections due to the extraordinary tolerance of biofilms to antimicrobial treatments. We have reason to believe that we have identified a biofilm-specific regulatory circuit required to induce the transition of biofilm bacteria from a susceptible to a drug resistant state. This pathway relies on the membrane-bound two-component sensor SagS and the MerR-like, c-di-GMP responsive transcriptional regulator BrlR (PA4878). BrlR activates the expression of several genes encoding proteins involved in multidrug efflux, thus, affecting MIC, susceptibility, and recalcitrance of P. aeruginosa biofilms o killing by bactericidal antimicrobial agents. SagS contributes to brlR expression and BrlR DNA binding in a manner dependent on c-di-GMP levels. Induction of brlR expression is linked to biofilm development, with transition to the irreversible attachment stage, regulated by SagS, marking the timing of biofilm cells gaining their heightened tolerance to antimicrobial agents. Our findings thus, suggested SagS to act as a molecular switch to control biofilm formation and tolerance. However, little is known about how this is accomplished by SagS. Understanding the mechanism underlying this switch, however, will enable us to manipulate the biofilm tolerance response, with the potential to impair the transition of biofilm bacteria from a susceptible to a resistant phenotype likely resulting in enhanced treatment options in fighting biofilm infections. The goal of this project is to elucidate SagS-dependent signaling and regulatory events contributing to biofilm cells transitioning to an antimicrobial tolerant state in a BrlR-dependent manner. The project is founded on the hypotheses that BrlR and SagS are linked via a signaling cascade, requiring in a sequential manner the action of SagS, in particular its periplasmic sensory HmsP domain, SagS-signaling to activate DGC PA3177, with PA3177 contributing to the biofilm c-di-GMP level which in turn is required to activate BrlR via c-di-GMP binding and thus, biofilm tolerance. Experimentally, we will determine in Aim 1 the contribution of the HmsP domain of SagS to its function as a molecular switch by determining residues or extended sequences within the HmsP domain, responsible for SagS contributing to in vitro and in vivo biofilm tolerance and biofilm formation, and interaction with BfiSR. In Aim 2, we will determine how SagS, in a manner dependent on input and signaling, contributes to DGC PA3177 to modulate biofilm c-di-GMP levels. In Aim 3, we will determine how PA3177 and c-di-GMP contribute to BrlR function, identify BrlR amino acid residues contributing to c-di-GMP binding, and determine whether impaired c-di-GMP binding by BrlR results in biofilm cells impaired in transitioning to an antimicrobial tolerant state.